Frequency of 677 C T and 1298 A C polymorphisms in the 5 , 10-methylenetetrahydrofolate reductase ( MTHFR ) gene in Turner syndrome individuals

Turner syndrome (TS) is an interesting model for investigating the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and non-disjunction because of the high frequency of chromosomal mosaicism among patients with this syndrome. We determined the frequencies of MTHFR 677C T and 1298A C polymorphic mutations in 49 patients with TS and 200 control individuals. The frequency of the 677C T allele was 0.39 for patients and 0.29 for controls while that of the 1298A C allele was 0.28 for patients and 0.25 for controls. Genotype frequencies were shown to be different in patients and controls ( 2 = 12.143; p = 0.033), and this was attributable to the higher frequency of the C677C T /677C T genotype among TS patients. In homozygotes, this mutation might have an effect on somatic chromosome disjunction by decreasing MTHFR activity.

Defects in folic acid metabolism because of mutations in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene have been considered as a possible cause of chromosomal non-disjunction by hypomethylation (James et al., 1999;Hobbs et al., 2000).The MTHFR gene is located on the short arm of chromosome 1 (1p36.3)and has 11 exons (Goyette et al., 1994) which code for a cytosolic flavoprotein that catalyzes the reduction of 5,10-methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methylTHF), the predominant circulating form of folic acid (Weisberg et al., 1998).One of the metabolic products, S-adenosylmethionine (SAM), is a methyl donor in various methylation reactions, including DNA and protein methylation and phospholipid synthesis (Chiang et al., 1996).Frosst et al. (1995) identified a C to T substitution at nucleotide 677 in the coding region of the MTHFR gene converting an alanine to a valine residue in the gene product and creating a new HinfI restriction site resulting in the MTHFR 677C ® T polymorphism and a reduction in enzyme activity which may lead to a decrease in the levels of SAM, inhibition of methyltransferase and subsequent DNA hypomethylation.The importance of the stable methylation of pericentromeric DNA for chromosomal stabilization and segregation has been supported both by clinical and experimental data (reviewed by Hobbs et al., 2000).Therefore, in homozygosis, the 677C ® T mutation may be a contributing risk factor to somatic chromosomal non-disjunction.James et al. (1999) studied the mothers of Down syndrome children and found a high frequency of mothers who were heterozygous for the MTHFR 677C ® T polymorphism, suggesting that this mutation might be a risk factor for chromosome 21 non-disjunction.Hobbs et al. (2000) further investigated this association and in addition studied the methionine synthase reductase (MTRR) gene, also involved in folate metabolism, and found not only that the MTRR 66A ® G polymorphism was independently associated with a 2.6 fold increase in the estimated risk for Down syndrome when mothers were homozygous for gene but that the presence of both the polymorphisms was associated with a greater risk than was the presence of either alone.
However, the association between the MTHFR 677C ® T and MTRR 66A ® G polymorphisms is still controversial.In an extensive study, Hassold et al. (2001) determined the frequencies of the MTHFR 677C ® T polymorphism in mothers of 93 cases of sex chromosome trisomy, 158 cases of chromosome 2, 7, 10, 13, 14, 15, 16 and 22 trisomy and 44 cases of trisomy 18 but detected significant increase in the 677C ® T polymorphism only in mothers of trisomy 18 concepti.Even so, Chadefaux-Vekeman et al. (2002) did not detect any significant differences in 667C ® T genotype frequencies among 85 mothers of fetuses with DS and 107 controls.A similar finding was reported by Petersen et al. (2001), who analyzed 177 mothers of DS patients resulting from maternal non-disjunction in meiosis I or II.
A possible explanation for these contradictory findings is the influence of maternal age, and it may be that MTHFR deficiency is more prevalent as a cause of nondisjunction among younger mothers of Down syndrome children while in older mothers maternal age may be the risk factor.In the study by Chadefaux-Vekemans et al. (2002) the mean age of the mothers of Down syndrome children was 34.9 years (range: 20 to 47 years), whereas in the study by James et al. (1999) the mean age was 29.7 years and all the mothers were less than 40 years old.
The A to C substitution at base pair 1298 of the MTHFR gene (van der Put et al., 1998) causes a glutamate to alanine substitution (the MTHFR 1298A ® C polymorphism) which abolishes the MboII restriction site created by the MTHFR 677C ® T polymorphism.
Turner syndrome individuals frequently show mosaicism (Hook and Warbuton, 1983;Robinson, 1990) and the presence of a normal cell lineage along with the 45,X lineage is considered to increase the probability of survival of those with 45,X Turner syndrome (Held et al., 1992, Kelly et al., 1992).
Our study was carried out because Turner syndrome can be used as a model for investigating the contribution of the MTHFR gene polymorphisms to somatic chromosomal non-disjunction and help to shed light on the role of this genetic mechanism in both Down and Turner syndrome.
The study group consisted of 49 Turner syndrome individuals (mean age 15.6 ± 6.4; range 3 to 33 years) of predominantly European descent.For each individual we assessed metaphases from a minimum of 50 and a maximum of 113 blood lymphocytes, 50 cells providing a probability of »99.9% of detecting a mosaicism in which the minority cell line occurs with a frequency of at least 10% or a probability of »96.2% of detecting a mosaicism including a minority cell line with a frequency of at least 5% (Bei-guelman, 1982).In our group 24 individuals had a 45,X karyotype and 25 had at least one additional cell line (i.e.22 individuals with 45,X/46,XX, one with 45,X/46,XX/ 47,XXX, another with 45,X/46,XY and a third with 45,X/ 46,XX/47,XX + 13).The control group was randomly selected from a group of blood donors in apparently good health and consisted of 106 females plus 94 males (n = 200 with a mean age = 27.3 ± 6.1 years; range 18 to 43 years), 110 being of predominantly European descent and 90 of predominantly African descent.This study was approved by the ethics committee of our institution and informed consent was obtained from each individual or their legal guardians.
Genomic DNA of members of the study and control groups was extracted from lymphocytes and purified (Woodhead et al., 1986) and the 677 region of the MTHFR gene PCR amplified using forward (5'-TGAAGGAGAAG GTGTCTGCGGGA-3') and reverse (5'-AGGCGGTGC GGTGAGAGTG-3') primers to generate a 198-bp fragment which was digested with HinfI to yield a 23 and 175 bp fragment that were separated by polyacrylamide gel electrophoresis (Figure 1).
In the 49 Turners syndrome individuals the frequency of the two alleles was 677C ® T = 0.39 and 1298A ® C = 0.28 while that in the 200 control individuals was 677C ® T = 0.29 and 1298A ® C = 0.25 (Table 1).Although the differences between the two groups were not significant by the chi-squared test (c 2 = 5.5; for 2 degrees of freedom (df) at p = 0.064) analysis of the adjusted residuals using Haberman's test suggested that the frequency of the normal allele was somewhat decreased in the Turner syn-  group.When the Turner syndrome individuals were grouped genotypically according to polymorphism a significant difference was observed for the 667C ® T polymorphism (c 2 = 8.822; df = 2; p = 0.012) but not for the 12198A ® C polymorphism (c 2 = 0.448; df = 2; p = 0.799).Subsequent analysis of all possible genotypes using both alleles (Table 2) revealed a significant difference between Turner syndrome individuals and controls (c 2 = 12.143; df = 5; p = 0.033), the 677C ® T/677C ® T homozygote genotype being more frequent among Turner syndrome individuals as shown by an analysis of adjusted residuals using Haberman's test.When individuals of African descent were excluded from the control group, the differences persisted (c 2 = 12.46; df = 5; p = 0.029).
Our results show an increase in the frequency of the MTHFR 677C ® T homozygote genotype in Turner syndrome individuals.Mosaicism among these patients is frequent and it is believed that the presence of a normal cell lineage along with the 45,X lineage confers viability to a conceptus (Kelly et al, 1992).Indeed, in our study about half of the Turner syndrome individuals tested were mosaics, implying post-zygotic non-disjunction events.As mentioned above, the 677C ® T polymorphism causes a reduction in enzyme activity (Frosst et al., 1995) which may lead to inhibition of methyltransferase through decreased levels of SAM and result in DNA hypomethylation.Both clinical and experimental data support the importance of pericentromeric DNA methylation for chromosomal stabilization and segregation (Hobbs et al., 2000) and it appears that in homozygosis the 677C ® T mutation may be a contributing risk factor to somatic chromosomal nondisjunction.

Table 1 -
Frequencies of the methylenetetrahydrofolate reductase (MTHFR) 677C ® T and 1298A ® C alleles in 49 individuals with Turner syndrome and 200 controls.

Table 2 -
Genotypic distribution of the methylenetetrahydrofolate reductase (MTHFR) 677C ® T and 1298A ® C alleles in 49 individuals with Turner syndrome and 200 controls.